A small molecule that induces assembly of a four way DNA junction at low temperature

Article abstract of DOI:10.1039/c1cc12922h

Small molecules that induce the formation of higher order DNA structures have potential therapeutic and nanotechnology applications. Screening of a click library has identified the first compound to induce the formation of a Holliday junction structure at

Full text of DOI:10.1039/c1cc12922h

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A small molecule that induces assembly of a four way DNA junction at
low temperaturew
a
a
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Lesley A. Howell, Zoe A. E. Waller, Richard Bowater, Maria O’Connell and
Mark Searcey*a
Received 18th May 2011, Accepted 9th June 2011
DOI: 10.1039/c1cc12922h
Small molecules that induce the formation of higher order DNA
structures have potential therapeutic and nanotechnology
applications. Screening of a click library has identified the first
compound to induce the formation of a Holliday junction
structure at room temperature without the need for a high
temperature annealing step.
The prototype HJ binding molecule from our previous
studies consisted of a symmetrical dimer of 9-aminoacridine-
4-carboxamide. We have previously shown that it is possible to
generate both 3- and 4-carboxamides using click chemistry
with an azidoethylamino-group at the 9-position of the
acridine.9 Compound 1 was made through similar click reactions
but this time utilizing a 9-propargylamino- substituent on the
acridine chromophore.10 Reactions of this type are somewhat
more limited due to the unstable nature of the starting acridine
substituted with an alkyne but compound 1 was isolated in
55% yield (Scheme 1).
In 1997, Hurley and Neidle described the inhibition of telomerase
through stabilization of a G-quadruplex in telomeric DNA
sequences with a small molecule.1 Since then, there has been an
explosion of interest in targeting higher order DNA structures,
particularly as G-rich sequences have been identified in the
promoter regions of oncogenes2 and targeting these structures
has been shown to modulate gene expresion.3 A number of
other higher order DNA structures also exist and have been
the subject of investigation.4 The most well studied structure is
the triple helix, in which a third strand of DNA or RNA binds
in the major groove and molecules have been designed with an
extended surface to target these by intercalation.5 DNA three
way junctions can be targeted by organometallic tube-like
structures and have been shown to have interesting biological
activity.6 We and others have demonstrated that small molecules
can also bind to the four way junction known as the Holliday
junction (HJ). Peptides that bind to the junction were originally
described by Segall and co-workers7 and may have potential as
anti-bacterials. We have shown that a compound that was
originally designed as a threading DNA bisintercalator can
reach across the junction and displace the adenine from an AT
base pair.8 This novel mode of binding prompted us to search
for other compounds that bind to a HJ and that may have
potential as biological probes for HJ structures in vivo or as
additives in nanotechnology applications. Herein, we describe
the first example of a molecule that promotes the formation of
a four way junction via both thermal annealing and at room
temperature.
Four oligonucleotides, labelled b, h, r and x (for sequences see
the ESIw) were designed to form an X-stacked four way junction
under standard annealing conditions in the presence of divalent
metal ions.11 In aqueous solution and following annealing in the
absence of divalent metal ions, the oligonucleotides exist as an
equilibrium mixture of single stranded DNA and the ‘‘open’’ form
of the junction (Fig. 1). On the addition of divalent metal
ions such as Mg2+ and high temperature annealing, the HJ is
converted to the X-stacked form.
With oligonucleotide
x doubly labelled with FAM
(6-carboxyfluorescein) and TAMRA (tetramethyl-6-carboxyrhod-
amine), it was possible to visualize the formation of the HJ on
a non-denaturing polyacrylamide gel following high temperature
annealing in the presence of increasing concentrations of
Mg2+ (Fig. 2a). It was clear that at a concentration of around
1 mM the HJ was formed with some small amount of single
stranded DNA remaining even at 5 mM concentration. When
the same oligonucleotides were annealed in the presence of
a School of Pharmacy, University of East Anglia,
Norwich Research Park, Norwich NR4 7TJ, UK.
E-mail: m.searcey@uea.ac.uk; Fax: +44-1603-592009;
Tel: +44-1603-492026
b School of Biological Sciences, University of East Anglia,
Norwich Research Park, Norwich NR4 7TJ, UK
w Electronic supplementary information (ESI) available: Experimental
details for the synthesis of 1 and for the assays described. See DOI:
10.1039/c1cc12922h
Scheme 1 Synthesis of compound 1 under click conditions.
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spectrum of HJ in the presence of 10 mM trisÁHCl, pH 7.00
shows a negative signal at 250 nm and a positive signal at
277 nm. The CD spectrum of HJ annealed in the presence of
10 mM trisÁHCl, pH 7.00 and 10 mM of MgCl2, however,
demonstrates more intense signals at 250 nm (negative) and
277 nm (positive), indicative of the spectral difference between
the open and closed HJ structures (Fig. 3a).12 HJ annealed in
10 mM trisÁHCl, pH 7.00 and 100 mM of ligand 1 (and no
added MgCl2) shows enhanced signals at À250 and +277 nm,
analogous to the effect observed when annealed in the presence
of MgCl2. This supports the results from the gel-based assay
and indicates that ligand 1 stabilizes the closed form of the
junction in a manner analogous to divalent metal ions. Taken
together, the evidence from the gels and CD experiments
indicate that ligand 1 promotes formation of the four way
junction during thermal annealing and does not require the
presence of divalent metal ions.
Fig. 1 Annealing in the presence of Mg2+ leads to the X-stacked
(or closed) form of the junction.
This led us to consider whether 1 could promote the
formation of four way junctions at room temperature without
a high-temperature annealing step. To explore this hypothesis,
we utilised CD titrations at room temperature. Compound 1
was titrated into pre-annealed solutions of HJ in the presence
of 10 mM trisÁHCl, pH 7.00, but in the absence of MgCl2
(i.e. in the open form), mixed thoroughly and a spectrum
acquired immediately. On titrating 1 into the solution of HJ, a
subsequent increase in ellipticity was observed at 250 nm
(negative) and 277 nm (positive), consistent with the effect
observed when the HJ sample was annealed in the presence of
Mg2+ (Fig. 3b). A similar effect was also observed when 1
was titrated into HJ annealed in the presence of 10 mM Mg2+
(see ESIw). We considered that the changes in ellipticity may
arise from 1 intercalating with the duplex ‘‘arms’’ of the
structure rather than at the junction itself. To rule this out
we repeated an analogous CD experiment with double helical
Fig. 2 Formation of the HJ detected by native polyacrylamide gel
electrophoresis. (a) PAGE FAM image showing the formation of the
HJ on addition of increasing concentrations of MgCl2 (for conditions
see ESIw). Lane C is a control lane where oligonucleotides b, h, r and x
are annealed in 450 mM NaCl, 24 mM Na citrate pH 7.0 and 2mM
MgCl2. (b) PAGE FAM image showing the effect of increasing
concentrations of compound 1 on the formation of the HJ. Lane C
as for (a).
compound 1, the HJ began to form at around 30 mM concen-
tration and there was no evidence of single stranded DNA
at a 50 mM concentration, suggesting that the compound
promoted formation of the HJ at least 100 fold more efficiently
than divalent metal ions (Fig. 2b).
An interesting observation in both gels is that at lower
concentrations both the divalent metal ions and compound 1
appeared to inhibit the formation of the HJ. This may be due
to the high affinity of both 1 and Mg2+ for the X-stacked form
of the four way junction and suggests that compound 1, at low
concentrations, prevents the formation of the open form.
Acridines are well known fluorescent molecules and we
utilized this property to probe binding dissociation constants
by fluorescence titrations with the same HJ oligonucleotides
without the fluorophores and with a double helical control
formed from oligonucleotide
x and its complementary
strand (see ESIw). Compound 1 showed moderately higher
affinity for HJ (Kd = 0.46 Æ 0.13 mM) compared to duplex
DNA (2.44 Æ 0.07 mM) and a stoichiometry plot indicated a
1 : 1 binding mode.
Fig. 3 CD spectra of HJ annealed in the presence of 10 mM trisÁHCl
pH 7.00 at 20 1C: (a) annealed in the absence of MgCl2 (black);
annealed in the presence of 10 mM of MgCl2 (blue); annealed in the
presence of 100 mM of ligand 1. (b) Titration with ligand 1 (0–150 mM).
To further explore the properties of 1 and its interaction
with the HJ we employed circular dichroism spectroscopy to
study whether 1 could affect the folding of the DNA. The CD
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sequences. Fluorescence and CD spectroscopy also demon-
strate that the molecule has a lower affinity for and different
effects on the structure of duplex DNA.
The biological potential of a molecule that promotes the
formation of the HJ is not known. Compound 1 is one of the
few 9-aminoacridine-3-carboxamide structures that we have
studied that has potent antitumour activity (9.1 mM in human
leukaemia HL60 cell line) although the modest difference in
affinity for the HJ versus duplex DNA may suggest that this
derives from classical topoisomerase inhibition rather than
effects on DNA repair processes. However, studies on the
ability of 1 and analogs to target other HJ forming sequences
and their effects on enzymes binding to four way junctions are
underway.
This research also highlights the potential of compounds
that can promote the formation of higher order DNA structures
in nanotechnology. It is likely that screening for compounds
with this ability will allow the formation of DNA nanostructures
without the requirement for a high temperature annealing step
and may change the physicochemical properties of the resulting
structures.
Fig. 4 Gels and densiometry plots showing the percentage of HJ
versus single stranded oligonucleotide over the designated time period.
(a) Oligonucleotides b, h, r and x were mixed together at room
temperature in the presence of 2.0 mM MgCl2 and left in the dark
for the specified time (final HJ concentration 1.25 mM). (b) Oligo-
nucleotides b, h, r and x were mixed together in the presence of 1
(50 mM) at room temperature and left in the dark for the specified time
(final HJ concentration 1.25 mM).
We thank Dr Myles Cheeseman of the Henry Wellcome
Laboratories for Biological Chemistry, UEA, for the use of the
CD spectrometer and Dr Nick Watmough of the School of
Biological Sciences, UEA, for the use of the fluorimeter.
LH was funded by a UEA studentship. We thank the Swansea
EPSRC Mass Spectrometry Service for high resolution mass
spectra.
DNA formed from x and its complementary sequence. Titration
of 1 with the double helical ‘‘control’’ resulted in no changes in
ellipticity (see ESIw), indicating the changes observed in the HJ
samples are the result of a specific interaction with the junction
as opposed to duplex intercalation. This is also consistent with
the 1 : 1 binding mode indicated by the stoichiometry plot.
Finally, we investigated the ability of the ligand to induce
the formation of a HJ without prior annealing using a gel
assay. Mixing the four oligonucleotides in solution at 20 1C in
the presence of 2 mM Mg2+ led to the formation of around
60% HJ over a 24 h period (Fig. 4a), demonstrating that even
in the presence of high concentrations of divalent metal ions, a
high temperature annealing step is required for the formation
of the HJ. Notably, after 6 h, around 40% of the four
way junction is present in solution. Carrying out the same
experiment with 50 mM of compound 1 in both the absence
and presence of Mg2+ ions (2 mM) led to the rapid formation
of the HJ structure even at 20 1C (Fig. 4b shows the experiment in
the presence of the divalent ions). Within 4 h all of the
oligonucleotides in solution had formed the four way junction.
The ability of a small molecule to promote the formation of
a four-way DNA junction is unprecedented. Within higher
order DNA studies, several groups have previously described
molecules that appear to promote the formation of a
G-quadruplex structure, although usually within a defined
intramolecular oligonucleotide sequence.13 Here, compound
1 is not just perturbing the equilibrium between the single strands
and open-X form of the junction but, from the CD data, is
promoting the formation of the X stacked form in a similar
way to divalent metal ions. This suggests that the compound is
promoting the intermolecular assembly of oligonucleotide
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8264 Chem. Commun., 2011, 47, 8262–8264
This journal is The Royal Society of Chemistry 2011